Cytotoxicity of momordin-folate conjugates in cultured human cells

We have shown previously that macromolecules can be nondestructively delivered into cultured cells via folate receptor-mediated endocytosis if the macromolecules are conjugated to folic acid prior to addition to receptor-bearing cells (Leamon, C.P., and Low, P. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5572-5576). Although an intracellular destination of the folate-linked proteins could be easily documented, the spatial resolution of the earlier data was insufficient to evaluate whether any endocytosed material was delivered into the cytosol. To resolve this issue, a folate-toxin conjugate was constructed using the impermeable ribosome-inactivating protein, momordin. Diminution of [3H]leucine incorporation into newly synthesized protein was then employed as a quantitative measure of the entry of the toxin into the cytosol. In studies with both HeLa and KB cells, cellular protein synthesis was found to be inhibited in a time- and concentration-dependent manner by the momordin-folate conjugate, but not by the underivatized toxin. IC50 values centered around 10(-9) M for the folate-linked samples. These observations provide direct evidence that folate conjugates not only reach the cytosol, but do so in a functionally active form.